Eukaryotic DNA replication is accomplished by initiating replication forks at multiple origins of replication in an ordered sequence during S phase. Replication defects are a major source of genomic instability leading to a variety of human disorders including cancer; in the yeast Saccharomyces cerevisiae, perturbing the orderly replication of the genome leads to genome instability. Cells have evolved elaborate surveillance mechanisms to monitor the integrity and orderly duplication of their genomes. To fully understand the causes and mechanisms of genome-instability disorders, therefore, it is crucial that we understand the molecular mechanisms choreographing the replication program and the mechanisms that ensure faithful genome maintenance in the face of DNA damage and stress. In examining some chromosomal rearrangements that have been reported in yeast, we note that in addition to the known repeated elements (such as tRNAs and transposons), there is anecdotal evidence for origins of replication very close to the breakpoint, leading us a to a new line of enquiry: to ask whether origins are intrinsically destabilizing elements in the genome. Drawing on evolutionary comparisons of S. cerevisiae with its distant, pre-genome- duplication relative Kluyveromyces waltii, we have found that origins in S. cerevisiae are indeed very highly correlated with genome rearrangement breakpoints. This proposal therefore has two broad themes: 7 A continuation of our ongoing efforts to understand the molecular mechanisms of origin action and regulation, with particular focus on how origins respond to replication stress and limiting replication factors. 7 Experimental tests for a direct link between the presence and activity of replication origins and genome instability. Because genome rearrangements are a major hallmark of cancer progression, we feel that these lines of enquiry will elucidate the interplay between origin function and genome stability, and in the long term have potential for developing both diagnostic and treatment strategies. PUBLIC HEALTH RELEVANCE: Perturbations in the orderly duplication of chromosomal DNA are a major source of genomic instability leading to a variety of human disorders including cancer. At the same time, there is evidence that origins of replication, the sites where DNA synthesis is initiated, are themselves potential agents of genome rearrangements. In this work, we propose to continue our investigations into the mechanisms that modulate the orderly progression of replication, and begin exploring the possible role of replication origins in leading to genome instability. This work will expand our understanding of the mechanisms underlying genome disorders, shed light on questions of health importance, and contribute to our ongoing efforts to understand the interplay between genome structure and replication.